Background: Isoprostane F 2␣ (iPF 2␣ -III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography-mass spectrometry (GC/MS) methods for measuring iPF 2␣ -III involve several steps of sample preparation and are labor-intensive, and ELISA methods, although easier to use, are less reliable. Therefore we developed a simple and sensitive method involving 1-step sample cleanup and HPLC/MS quantification. Methods: Samples of plasma or urine were enriched with a deuterated (iPF 2␣ -III-D4) standard, treated with KOH to liberate the bound isoprostanes, then loaded onto an immunoaffinity column, and the bound isoprostane was eluted with 95% ethanol. The concentrated sample was injected onto a C-18 HPLC column, and the isoprostane was eluted with a gradient of acetonitrile in water and analyzed by electrospray negative ionization, selectively monitoring the ions 353.2 (iPF 2␣ -III) and 357.2 (iPF 2␣ -III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions. Results: The described method has a detection limit of 0.5 ng/L, with a linear dynamic range of 1-5000 ng/L. The intra-and interassay imprecisions were 4.68% and 3.88%, respectively. The values obtained correlated strongly with the GC/MS procedure (r ‫؍‬ 0.80), but the absolute values were ϳ4 -to 5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together.

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans10 cis12 (A), trans9 trans11 (B), cis9 trans11 (C), and cis9 cis11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.

To assess the antioxidant behavior of trans-1, we first synthesized trans-allyl ether 4 by opening an (S)-glycidol derivative with an (E)-alk-2-en-ol, and then produced the unnatural E-enol ether 1 by a stereoselective iridium(I)-catalyzed olefin isomerization. Natural cis-1 was preferentially degraded by HOCl and was more protective than trans-1 against lipid peroxidation induced by a free-radical initiator, demonstrating that the geometry of the 1’-alkenyloxy bond participates in the antioxidant defensive role of 1.

No abstract